| 1. |
- Abouzayed, Ayman, 1992-
(författare)
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Theranostic Targeting of GRPR and PSMA in Prostate Cancer
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is based on five original articles that investigated the theranostics of prostate cancer by gastrin-releasing peptide receptor (GRPR) and prostate-specific membrane antigen (PSMA) targeting. GRPR and PSMA are two extensively evaluated prostate cancer cell markers due to their overexpression in the majority of prostate cancer samples. Theranostic targeting of GRPR and PSMA is an attractive strategy to improve the management of prostate cancer patients.Papers I and II focused on the dual targeting of GRPR and PSMA. The effect of linker modification on the affinity for GRPR and PSMA and the pharmacokinetic profile was evaluated. In Paper III, the effect of the GRPR antagonist RM26 conjugation to an albumin-binding domain on the pharmacokinetic profile and its potential use in therapy was investigated. Paper IV focused on developing a GRPR antagonist that was suitable for single-photon emission computed tomography (SPECT) using technetium-99m. In Paper V, the GRPR antagonist developed in Paper IV was translated into a phase I clinical trial to assess safety and dosimetry.Modifying the linkers in GRPR and PSMA heterodimers can largely impact the affinity for both targets. This modification influenced the in vivo targeting specificity and biodistribution, with [125I]I-BO530 in Paper I and [111In]In-BQ7812 in Paper II outperforming other analogues. Our findings in Paper III indicated that the conjugation of an albumin-binding domain to RM26 increased the blood concentration of the radiotracer. This increase led to elevated and stable tumour uptake of [111In]In-DOTA-ABD-RM26 after several days of injection. However, [111In]In-DOTA-ABD-RM26 was also increasingly taken up by various healthy organs. The GRPR antagonist [99mTc]Tc-maSSS-PEG2-RM26, studied in Paper IV, showed high specificity and affinity for GRPR. This resulted in elevated GRPR-mediated uptake. Additionally, maSSS-PEG2-RM26 could be radiolabelled via a straightforward radiolabelling protocol. Clinical evaluation of [99mTc]Tc-maSSS-PEG2-RM26 in prostate and breast cancer patients (Paper V) demonstrated the safety and tolerability of the radiotracer, with favourable dosimetry and no side effects.In conclusion, this thesis evaluated different tools for the theranostic targeting of GRPR and PSMA. The findings warrant further investigation to optimise the reported radiotracers.
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| 2. |
- Aboye, Teshome Leta, 1975-
(författare)
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Engineering of the Ultra-stable Cystine Knot Framework of Microproteins : Design, Chemical Synthesis and Structural Studies
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ultra-stable cystine knotted microproteins, in which two disulfides and their connecting backbones form a circle that is penetrated by the third disulfide bonds, have attracted high interest due to their resistance to degradation in vitro and potential for the development of peptide drugs. This thesis gives new insights into engineering of that framework of microproteins, including approaches to their chemical synthesis, backbone engineering, structural and biological evaluations. Synthetic and oxidative folding approaches for bracelet cyclotides, a family of cyclic cystine knotted microproteins, was developed using a model peptide, cycloviolacin O2. Following assembly of the peptide chain, protected peptide was generated by mild cleavage that was subsequently thioesterified and cyclized in solution. The cyclic peptide was oxidatively folded under optimized conditions containing co-solvent and non-ionic detergent affording native cycloviolacin O2 as a major product. To gain further insights into the heterogeneity, efficiency and kinetics of cyclotides’ oxidative folding, the intermediates that accumulate in oxidative refolding pathways of all cyclotide subfamilies: Möbius, bracelet and the hybrid cyclotides were quantitatively determined under four different folding conditions. The results were used for defining major folding pathways, which indicated that Möbius cyclotides might accumulate heterogeneous folding intermediates with one-, two- and three-disulfides, whereas bracelet tend to accumulate a homogenous intermediate with three-disulfides, depending on the buffer systems used. Furthermore, to probe the internal factors contributing to inefficiency of oxidative folding, as well as undesired bioactivities of bracelet cyclotides (e.g., cytotoxic activity), polymer-hybridized cyclotides were designed by replacing non-conserved residues with small isosteric polymers. The designed hybrid analogs in which hybridization involved replacement of loop 3 with isosteric polymers showed improved synthetic and oxidative folding properties. The cytoxicity of a model hybrid designed with replacement of loop 3 and 5 exhibited no cytotoxic activity at concentration of 128-fold relative to that of native peptide. Furthermore, 1D and 2D 1H NMR analysis of this hybrid showed that it had well structured fold.
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| 3. |
- Abrantes, João A.
(författare)
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Pharmacometric Approaches to Improve Dose Individualization Methods in Hemophilia A
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hemophilia A is a bleeding disorder caused by the lack of functional coagulation factor VIII (FVIII). The overall aim of this thesis was to improve dose individualization of FVIII replacement therapy in hemophilia A using pharmacometric approaches.A population pharmacokinetic (PK) model of FVIII activity following the administration of moroctocog alfa was developed based on data from a large heterogeneous cohort of moderate to severe hemophilia A patients. Body weight, age, neutralizing anti-FVIII inhibitors, race, and analytical assay were found to be significant predictors of FVIII activity PK. In addition, large inter-individual variability (IIV) and inter-occasion variability (IOV) was identified highlighting the need for dose individualization.High magnitudes of IOV are known to impair model-based therapeutic drug monitoring. Using a population PK model of FVIII activity, several approaches to handle IOV in Bayesian forecasting of individual PK parameters were assessed across a wide range of features. Considering IOV in Bayesian forecasting, but ignoring IOV in dose calculation, led to the most precise individualized doses, in particular, when sparse data was used.The dose-exposure-response relationship of FVIII replacement therapy remains unclear. A parametric repeated time-to-categorical event (RTTCE) model was developed to characterize the relationship between the dose of octocog alfa, plasma FVIII activity, bleeding frequency and severity, and covariates, using data from clinical trials. The bleeding hazard was found to decrease throughout time and to be affected by plasma FVIII activity and number of previous bleeds. Unexplained IIV in the bleeding hazard was found to be large.Bayesian forecasting based on the RTTCE model was used to predict the future occurrence of bleeds, and to contrast the predicted outcome using individual i) PK, ii) bleeding, and iii) PK, bleeding and covariate information, from data collected in clinical trials. The results support that individual bleed information can inform the optimization of prophylactic dosing regimens in severe hemophilia A patients.In summary, the pharmacometric approaches presented provide a valuable quantitative framework to improve dose individualization in hemophilia A. Furthermore, enhanced dosing has the potential to reduce bleeding frequency and to lower the high costs associated to treatment.
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| 4. |
- Adeyemi, Ahmed, 1986-
(författare)
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Palladium(0)-Catalyzed Synthesis of Spirocycles and Supercritical Chemistry using a Resistively Heated Flow Reactor
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This doctoral thesis focusses on an effective and selective approach to the synthesis of spirocycles using palladium(0)-catalyzed Mizoroki-Heck reactions. In addition, selective and efficient chemistry was highlighted by the design and evaluation of a novel resistively heated system for continuous flow (CF) synthesis for high-temperature and high-pressure applications.Paper I described the design and evaluation of a novel resistively heated CF system. The design of a low-cost, simple, robust, and effective CF system involving a resistively heated steel reactor capable of delivering 400 °C and 200 bar was reported. The reactor was evaluated with esterification, transesterification and direct carboxylic acid to nitrile conversions using supercritical ethanol, methanol and acetonitrile respectively. Diels-Alder reactions under neat conditions were also carried out at high temperature and pressure.Paper II reported the synthesis of spirooxindoles by a selective application of the palladium(0)-catalyzed Mizoroki-Heck spirocyclization. The precursors for the reaction were synthesized by coupling 2-iodoanilines with esters derived from enantiomerically pure (+)-Vince lactam decorated with the bulky, directing 2,5-dimethylpyrrole protecting group. Ten different spirooxindoles were reported with good yields and high regio- and stereoselectivity. Functionalization of a synthesized spirooxindole was done by a palladium(0)-catalyzed alkoxycarbonylation, followed by selective deprotections.In Paper III, ether precursors were synthesized from (+)-Vince lactam, via a Mitsunobu reaction with the corresponding iodophenols. The precursors were later subjected to conditions for intramolecular Mizoroki-Heck reaction. Overall, 12 spiroethers were synthesized in useable yields, regioselectivity up to 98% and with excellent diastereoselectivity (d.e.>98%). Further functionalization to mono-protected rigidified amino acids was also demonstrated.
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| 5. |
- Aerts, Jordan
(författare)
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Capillary electrophoresis mass spectrometry applied to structural proteomics and small molecule analysis
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Capillary electrophoresis with mass spectrometric (CE–MS) detection offers a separation method without equal in terms of flexibility, utility, and cost efficiency. Here we demonstrate precisely this through the application of several laboratory-built CE–MS instruments for the separation of brain metabolites in non-primates, enantioselective separations of synthetic anesthetic metabolites in fractionated pony urine, application in structural proteomics workflows, and identification of exogenous alkaloid biotransformationproducts in human cerebrospinal fluid (CSF).We outline a method for quickly and affordably etching austenitic steel tubing, which is widely used in electrospray sources for CE–MS. The stainless steel tapered tip emitters provide robust electrospray with low sheath liquid flow rates and can be easily fabricated in-house, offering flexibility and cost-efficiency when commercial options areunavailable. We contribute a CE–MS method for enantiomer separation, specifically targeting 6-hydroxynorketamine (HNK). By introducing chiral selectors into the separation capillary, the method enables efficient enantiomer separation and offers a newtool to assist with research on HNK as a cure for depression.We explore the feasibility of cold CE–MS in hydrogen deuterium exchange workflows. The utilization of a lab-designed Peltier-cooled CE device achieves deuterium back exchange rates on par with commercial liquid chromatography-based platforms, offering new possibilities for studying protein structures and interactions.We also demonstrate the wide ranging versatility of CE–MS with contributions to the identification of specific tobacco related metabolites in CSF samples during the development of a high throughput mass spectrometry diagnostic tool for Parkinson’sDisease.This thesis showcases the versatility and value of CE–MS in various applications, a true blessing for analytical chemistry.
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| 6. |
- Ahlin, Gustav, 1977-
(författare)
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In vitro and in silico prediction of drug-drug interactions with transport proteins
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Drug transport across cells and cell membranes in the human body is crucial for the pharmacological effect of drugs. Active transport governed by transport proteins plays an important role in this process. A vast number of transport proteins with a wide tissue distribution have been identified during the last 15 years. Several important examples of their role in drug disposition and drug-drug interactions have been described to date. Investigation of drug-drug interactions at the transport protein level are therefore of increasing interest to the academic, industrial and regulatory research communities. The gene expression of transport proteins involved in drug transport was investigated in the jejunum, liver, kidney and colon to better understand their influence on the ADMET properties of drugs. In addition, the gene and protein expression of transport proteins in cell lines, widely used for predictions of drug transport and metabolism, was examined. The substrate and inhibitor heterogeneity of many transport proteins makes it difficult to foresee whether the transport proteins will cause drug-drug interactions. Therefore, in vitro assays for OCT1 and OATP1B1, among the highest expressed transport proteins in human liver, were developed to allow investigation of the inhibitory patterns of these proteins. These assays were used to investigate two data sets, consisting of 191 and 135 registered drugs and drug-like molecules for the inhibition of OCT1 and OATP1B1, respectively. Numerous new inhibitors of the transport proteins were identified in the data sets and the properties governing inhibition were determined. Further, antidepressant drugs and statins displayed strong inhibition of OCT1 and OATP1B1, respectively. The inhibition data was used to develop predictive in silico models for each of the two transport proteins. The highly polymorphic nature of some transport proteins has been shown to affect drug response and may lead to an increased risk of drug-drug interactions, and therefore, the OCT1 in vitro assay was used to study the effect of common genetic variants of OCT1 on drug inhibition and drug-drug interactions. The results indicated that OCT1 variants with reduced function were more susceptible to inhibition. Further, a drug-drug interaction of potential clinical significance in the genetic OCT1 variant M420del was proposed. In summary, gene expression of transport proteins was investigated in human tissues and cell lines. In vitro assays for two of the highest expressed liver transport proteins were used to identify previously unknown SLC transport protein inhibitors and to develop predictive in silico models, which may detect previously known drug-drug interactions and enable new ones to be identified at the transport protein level. In addition, the effect of genetic variation on inhibition of the OCT1 was investigated.
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| 7. |
- Ahnfelt, Emelie
(författare)
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In vitro evaluation of formulations used in the treatment of hepatocellular carcinoma
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hepatocellular carcinoma (HCC) causes ~ 600,000 deaths annually, making it the second most deadly cancer form. HCC is classified into five stages and for the intermediate HCC treatment, the two most commonly used drug delivery systems (DDSs) are lipiodol-based emulsions and drug-eluting beads. The aims of this thesis were to develop in vitro methods suitable for studying these DDSs. It is important to investigate the release mechanisms and release rates with relevant in vitro methods, as this can improve the understanding of the in vivo performance. Miniaturized in vitro methods with sample reservoirs separated from the release medium by a diffusion barrier were developed and shown to be suitable for studying drug release from particle DDSs (Paper I). In Paper II these methods were further developed and used to study the release of doxorubicin (DOX) from the clinically used drug-eluting beads. DOX release rates were affected by the method set-up and the characteristics of the release medium. The choice of method and volume of release medium could improve the in vivo-likeness of the in vitro release profiles. Applied theoretical models suggested a film-controlled type of DOX release mechanism from the beads when self-aggregation, DOX-bead interaction, and DOX deprotonation were taken into account.A micropipette-assisted microscopy method was used to further improve the understanding of the release mechanism of amphiphilic molecules from the beads (Paper III). A detailed analysis suggested an internal depletion-layer model dependent on molecular self-aggregation for the release. It was further suggested that a simple ion-exchange mechanism is unrealistic in physiological conditions.The important pharmaceutical factors for the emulsion-based formulations were investigated in Paper IV. DOX solubility, lipid phase distribution, and emulsion stability increased when the contrast agent iohexol was added. Also, an increase in release half-life (h) was observed from emulsions with iohexol.The in vitro methods and theoretical models presented in this thesis can be used during development and optimization of future DDSs.
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| 8. |
- Al Haj, Mahmoud
(författare)
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Effects of Dehydration and Blockade of the Renin-Angiotensin System in the One-humped Camel (Camelus dromedarius)
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The one-humped or the dromedarian camel is a pseudo-ruminant mammal, well adapted to the hot and dry climates of the desert. Its ability to withstand torrid heat and extreme desiccation is of paramount importance to its survival. The studies presented in this thesis were designed to investigate and document the effect of dehydration in the presence or absence of angiotensin II (Ang II) AT1 receptor blocker (losartan) on blood constituents, electrolytes, hormones, neurotransmitters as well as liver and kidney enzymes in a subset of dehydrated camels and to compare them with hydrated camels. Additionally, we studied the response of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) and revealed for the first time the cardiac storage form of BNP in the camel heart. Dehydration induced significant increments in packed cell volume (PCV), white blood cells (WBC), gamma glutamyl-transferase (GGT), serum sodium, creatinine and urea levels, and a doubling in plasma cortisol and arginine vasopressin (AVP) levels. At the same time dehydration caused significant decrease in body weights, plasma insulin like growth factor-1 (IGF-1) and its binding protein-3 (IGFBP-3), and a 50% decrement in ANP and BNP levels. Moreover, dehydration with and without losartan resulted in significant changes in stress hormones and anti-oxidants in plasma, liver and kidney homogenates. Losartan on one hand enhanced the effect of dehydration resulting in significant increases in sodium, creatinine and urea levels. In addition losartan raised the binding affinity of Ang II AT2 receptors in the small intestine with 8-fold and with 16-fold for liver AT1 receptors, indicating that Ang II AT1 and AT2 receptor binding sites were present in camel's small intestine while only AT1 receptor binding sites were found in the camel liver. One the other hand losartan resulted in significant decrease in body weights impaired the rise in anti-diuretic hormone and reduced aldosterone level. Finally, we showed that the proBNP is the storage form of BNP in the camel heart.
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| 9. |
- Al-Tikriti, Yassir
(författare)
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Microgels as drug delivery vehicles : loading and release of amphiphilic drugs
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Polyelectrolyte microgels are used as delivery vehicles for amphiphilic drugs in, e.g., treatments of liver cancer by a method called trans-arterial chemoembolization. The thesis deals with fundamental properties of such delivery systems related to the self-assembling properties of the drug molecules and their interaction with the charged polymer network of the microgel. The main objective was to establish mechanistic models describing the loading and release of drugs under relevant conditions. For that purpose experimental techniques providing thermodynamic, compositional and microstructural information were used to elucidate how the kinetics depend on the stability of the drug self-assemblies and the volume response of the microgels. Micromanipulator-assisted microscopy studies showed that negatively charged microgels phase separated during loading and release of cationic amphiphilic drugs. At intermediate loading levels the drug aggregates and part of the network formed a collapsed phase coexisting with a swollen, drug-lean phase. In particular, during release in a medium of physiological ionic strength, the drug-lean phase formed a depletion layer (shell) surrounding a drug-rich core. Investigations of a series of drugs with different molecular architectures showed that the drug release rate was determined mainly by the stability of the drug aggregates in the core and the diffusive mass transport of drug molecules through the shell. Detailed studies of polyacrylate microgels interacting with amitriptyline hydrochloride showed that swelling of the shell network greatly influenced the release rate. Furthermore, experiments with a specially constructed microscopy cell was used to establish that the collapsed and swollen phases could coexist in equilibrium, and that the swelling of the network in the swollen phase depended on the proportion between them when present in the same microgel. The latter effect was related to the elastic coupling between the phases. Confocal Raman microscopy was employed to demonstrate, for the first time, the related elastic effect, that the concentration of amitriptyline in the swollen phase decreased with increasing proportion of the collapsed phase. Small-angle X-ray scattering showed that the collapsed phase had a disordered microstructure of drug micelles with ellipsoidal shape. The aggregation number increased with increasing concentration of drug in the microgel, most likely by incorporating the uncharged base form. By providing detailed information about thermodynamic properties and microstructures, the results of the thesis provide a basis for rational design of microgel drug delivery systems.
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| 10. |
- Alenius, Malin, 1974-
(författare)
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Treatment Response in Psychotic Patients in a Naturalistic Setting : Classification, Genes, Drugs, Insight and Social Networks
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many patients with psychotic symptoms respond poorly to treatment. Various approaches have been made to classify these patients according to treatment response. However, existing classifications have been criticized for various reasons and a new classification system is needed. Further, no satisfactory explanation of the poor treatment response has been apparent. The general aim of this thesis was therefore to develop and validate a new classification method of functional remission in a naturalistic population of patients with psychosis and to utilize this classification to investigate the population from genetic, drug treatment, insight and social network points of view. Data for this cross-sectional study of patients (n=123) attending the Psychosis Outpatient Care clinic in the county of Jönköping, Sweden, were obtained from patient interviews, blood samples and information from patient files. The new classification method CANSEPT, which combines the CAN rating scale (CAN), the UKU side effect rating scale (SE) and the patient’s previous treatment history (PT), showed validity in discriminating the patients and was accepted well by the patients. CANSEPT was used to group the patients in the other studies in this thesis. The results indicated that the gene polymorphism ABCB1 3435T, was related to worse significant social and clinical needs for patients on olanzapine, while the polymorphism DRD2 Taq1 A1 was related to a greater risk of significant side effects; especially if male, or taking strong dopamine D2-receptor antagonistic drugs. Drug treatment factors were also related to treatment response; longer duration of untreated prodromal and early psychosis was seen for patients with current significant social and clinical needs and non-adherence to treatment was associated with worse significant side effects. Worse treatment outcomes also appeared to be associated with smaller social network groups, worse insight into illness, poorer knowledge of warning signs and worse coping strategies. In summary, CANSEPT was shown to be a useful valid, multidimensional tool for classification of treatment response. Gene polymorphisms, duration of untreated illness, non-adherence to treatment, social networks and knowledge should be taken into consideration when investigating inadequate treatment response.
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